Antioxidant composition comprising acetyl L-carnitine and α-lipoic acid

ABSTRACT

A composition containing the active ingredients acetyl L-carnitine and α-lipoic acid for the prevention and/or therapeutic treatment of various alterations and pathological states induced by free radicals, that may be in the form of a dietary supplement, dietetic support or of an actual medicine.

This is A 371 of PCT/1T99/00268 filed Aug. 19, 1999.

The present invention relates to a composition for the prevention and/ortreatment of tissular diseases brought about by the presence of freeradicals due to environmental pollution; brain or myocardial damagesinduced by free radicals following cerebral or myocardial ischaemia andattendant riperfusion; of the toxic or diabetic neuropathies and ofmetabolic disorders in the glucose utilization.

Accordingly, the composition may take the form and exert the action of adietary supplement or of an actual medicine, depending upon the supportor preventive action, or the strictly therapeutic action, which thecomposition is intended to exert in relation to the particularindividuals it is to be used in.

More particularly the present invention relates to an orally,parenterally, rectally or transdermally administrable composition whichcomprises in combination:

(a) acetyl L-carnitine or a pharmacologically acceptable salt thereof,optionally in combination with at least another “carnitine” where for“carnitine” is intended L-carnitine or an alkanoyl L-carnitine selectedfrom the group comprising propionyl L-carnitine, valeryl L-carnitine,isovaleryl L-carnitine or their pharmacologically acceptable salts; and

(1) α-lipoic acid.

A systemic deficiency of alkanoyl L-carnitines (ubiquitousnaturally-occurring compounds, the greatest concentrations of which areto be found above all in skeletal muscle and in the myocardium) is knownto lead to muscular and functional deficits which can be restored tonormal by the exogenous administration of these compounds.

The presence of acetyl L-carnitine has been ascertained both at cerebrallevel and in peripheral nervous tissue where its presence is necessaryfor normal nerve conduction.

The production of energy by carnitines occurs via intramitochondrialβ-oxidation of fatty acids, as well as via the oxidation ofbranched-chain amino acids and regulation of insulin activity.

Important for the purposes of the characterization of the biologicalactivity of carnitines are studies indicating their stabilising effectson cellular phospholipid membranes and on the integrity anddeformability of erythrocytes.

Acetyl L-carnitine in particular protects cerebral tissue againstperoxidative phenomena. While it has been ascertained that carnitine isnecessary for normal growth, it is equally true that reduced carnitinelevels compared to normal have been detected during ageing.

During the metabolic processes associated with ageing, an increase inoxidative processes is constantly detected together with a relatedincrease in free radicals, which facilitates the onset of diabeticlesions.

Reduced mitochondrial activity leads to an increase in oxidants whichthe cell defences are no longer able to combat effectively.

The increase in peroxides, hydroxides and singlet oxygen produced byaerobic metabolism may lead to damage to macromolecules (DNA, proteinsand lipids), which contributes to the onset of degenerative diseases,including diabetes, which usually arise during ageing. The reducedmitochondrial activity which comes about with ageing is also accompaniedby a reduction in cardiolipin, a diphosphatyl-glycerol derivative whichmakes up part of the structure of the mitochondrial membrane and playsan important role in maintaining mitochondrial activity, particularly atthe level of fatty acid β-oxidation processes. Mitochondrial activity,including the fatty acid β-oxidation processes, can be reactivated bythe administration of acetyl L-carnitine, which is also capable ofrestoring normal cardiolipin concentrations in the mitochondria.

The positive effect of acetyl L-carnitine on mitochondrial activity isalso proved by its ability to promote the utilization of the glycolyticpathway for ATP production. These effects are detected particularly atthe neuronal level where acetyl L-carnitine has proved capable ofpreventing neuronal lesions or chronic neuronal degeneration.

In addition to a reduction in the carnitines present in the body duringthe processes of ageing, a reduction in growth factors (GF-I) is alsodetected and particularly a reduction in IGF-I (insulin-like growthfactor).

IGF-I, IGF-II and relaxin are peptides belonging to the group ofproinsulins also called somatomedins.

IGFs exert a homeostatic and trophic action, particularly at bothcentral and peripheral nervous system level, and the clinical use ofthese peptides has yielded beneficial results in many degenerativenervous disorders including diabetic neuropathy.

The correlations existing between ageing and a reduction in carnitinesand growth factors, including IGF-I, and the restoration of the levelsof these factors by means of the exogenous administration of acetylL-carnitine justify the interest in carnitines for the purposes of theiruse in the prevention and treatment of neurodegenerative diseases,including diabetic neuropathy.

It has been demonstrated that α-lipoic acid also performs an importantregulatory function on carbohydrate metabolism and insulin activity.α-lipoic acid is widely distributed in nature in both the vegetable andanimal worlds and can be taken with food. Recognised first as a growthfactor for a number of micro-organisms, it was then isolated in ox liveras bound to many animal proteins. It acts as an important scavenger offree radicals, above all those deriving from environmentalcontamination. Recently, it has been shown that this compound is alsouseful in the regulation of glucose utilization and of insulin activity,so much so as to constitute an important factor in the prevention ofdiabetic neuropathies.

It has been demonstrated that lipid peroxidation, which is increased indiabetic neuropathy, can be controlled and reduced, both at cerebrallevel and at the level of the sciatic nerve or the ocular lens, by theadministration of α-lipoic acid or one of its enantiomers. Moreover,α-lipoic acid inhibits the aldose reductase activated by hyperglycaemia,and therefore α-lipoic acid may also play an important therapeutic rolein diabetic complications.

α-lipoic acid enhances the insulin-induced muscular utilisation ofglucose and, in diabetic subjects, reduces resistance to the effects ofinsulin on glucose. Related to the antioxidant effect of α-lipoic acidare also its neuro-protective capability against brain damage induced byischaemia and its postulated therapeutic role in Parkinson's disease andAIDS.

The antioxidant effect of α-lipoic acid may be either direct orindirect, via restoration of glutathione and ascorbic acidconcentrations.

While the action of α-lipoic acid on carbohydrate metabolism is dueessentially to its ability to act as a coenzyme in the oxidativedecarbohydroxylation of pyruvate and other α-ketoacids and, through theacetates, in the activation of the tricarboxylic acid cycle leading tothe formation of ATP, for the purposes of explaining the multiplefavourable biological effects that this compound has in preventingdiabetic damage, other pathways whereby α-lipoic acid exerts itsprotective action should also be borne in mind.

Among these, one should bear in mind particularly the mechanismconsisting in its ability, after reduction to dihydrolipoic acid, toinhibit the activation of the nuclear transcription factor (NF-kB) byreactive oxygen species (ROS), thus, in turn, inhibiting the associatedcascade of neurotoxic and cytotoxic factors.

Since many of the complications associated with diabetes, such asneuropathies and ocular cataracts are mediated by ROS, inhibition ofactivation of the nuclear transcription factor may constitute amechanism via which α-lipoic acid may intervene in the prevention ofdiseases related to diabetes. Furthermore, one should also bear in mindthat, in diabetic subjects, the concentrations of α-lipoic acid arelower than normal values and that the administration of α-lipoic acidmay restore these levels to normal. It thus has an additive effect tothat of insulin in glucose transport to the cell membranes.

Chronic exposure to high concentrations of glucose may lead to anon-enzymatic reaction between glucose and proteins and to thespontaneous formation of highly reactive proteins known as end productsof glycosylation (Advanced Glycosylation End Products=AGEs). Amongthese, the gylcosylation products of glucose and albumin, glucose andcollagen, and glucose and haemoglobin are those most studied. Theeffects that AGEs give rise to in tissues and cells are all relevantfactors in explaining a large proportion of diabetic diseases atnervous, muscular and endothelial level.

AGEs, in fact, enhance the synthesis of the components of theextracellular matrix, increase endothelial permeability and theformation of immune complexes and cytokines, and cause neuronal andretinal ischaemia, myelin accumulation and myelinic degeneration. Anumber of these compounds are formed both in the course of diabetes andduring ageing.

A correlation between AGEs and activation of NF-IKB has recently beendemonstrated, as has the ability of x-lipoic acid to inhibit thisreaction.

Protein glycation and glucose oxidation by glucose at highconcentrations together with free radicals may, therefore, be another ofthe causes responsible for the tissue abnormalities—particularly nervetissue abnormalities—associated with diabetes. The presence of α-lipoicacid also inhibits or limits the progression of glycosylation or glucoseoxidation reactions.

Another protective effect of α-lipoic acid has also been observed inpancreatic cells placed in contact with inflammatory agents.

As regards the action of α-lipoic acid in the prevention and cure ofcataracts, this may be attributable not only to the other mechanismsdescribed above, but also to restoration of vitamin C concentrations inthe eye which are reduced by hyperglycaemia due to competition ofglucose with vitamin C transport.

In addition to a sparing of vitamin E and an increase in glutathioneconcentrations, the protective action of α-lipoic acid against the onsetof neuropathies has also been confirmed by clinical studies. It has beenobserved that the reduction in nervous lesions is also accompanied by areduction in peroxidative reaction products as detected by the loweringof malonylaldehyde concentrations. Other multicentre studies haveconfirmed its activity in the treatment of diabetic neuropathies.

Surprisingly it has been found that a composition comprising as itscharacterising components a combination of:

(a) acetyl L-carnitine or a pharmacologically acceptable salts thereof,and

(b) α-lipoic acid

is extremely effective in the prevention and/or treatment of tissuedamage induced by the presence of free radicals due to environmentalpollution; of cerebral or myocardial lesions induced by free radicalsafter cerebral or myocardial ischaemia and as a result of reperfusion;of toxic or diabetic neuropathies, and of metabolic disorders in theglucose utilization.

It has also been found that, advantageously, component (a) may furthercomprise a “carnitine” selected from the group comprising L-carnitine,propionyl L-carnitine, valeryl L-carnitine and isovaleryl L-carnitine ortheir pharmacologically acceptable salts thereof,

The (a):(b) weight-to-weight ratio ranges from 100:1 to 1:10.

Toxicological Tests

Both carnitines and α-lipoic acid are well known for their very limitedtoxicity and good tolerability. These favourable toxicologicalcharacteristics of carnitines and α-lipoic acid have been confirmed bycombining these components and administering them at high doses both torats and mice. In these animals, in fact, it proved possible toadminister amounts of up to more than 250 mg/kg of acetyl L-carnitine or100 mg/kg of α-lipoic acid parenterally, as well as of 250 mg/kg of amixture of carnitines (acetyl L-carnitine, propionyl L-carnitine,isovaleryl L-carnitine combined in a 1:1 weight ratio to one another)and more than 500 mg/kg of acetyl L-carnitine, 500 mg/kg of thecarnitine mixture and 200 mg/kg of α-lipoic acid orally without any ofthe animals thus treated dying.

Also prolonged administration via the diet for 30 consecutive days, bothin a group of rats and in a group of mice, of 200 mg/kg of acetylL-carnitine or 200 mg/kg of the carnitine mixture together with 100mg/kg of α-lipoic acid proved to be well tolerated and led to thedetection of no signs of toxicity. Both the weight gain and the variousblood-chemistry tests performed in these animals showed normal values,as did the findings of histopathology tests performed on the main organsafter sacrificing the animals at the end of treatment.

Neuroprotective Activity Tests in Experimental Cerebral Ischaemia

In view of the fact that lesions due to cerebral ischaemia are relatedto the production of free radicals and of nitrous oxide and that bothcarnitines and α-lipoic acid afford protection against the toxic actionof free radicals, in these tests cerebral ischaemia was induced byoccluding the middle cerebral artery (MCA) according to the methoddescribed by Scharkey (Scharkey, Y., Nature 371-336, 1994) by injectingendothelin-1 (120 pmol in 3 nl) into the anaesthetised rat in threeminutes with a microcannula placed stereotactically in the piriformcortex at the level of the middle cerebral artery. Occlusion of theartery is induced, and the resulting ischaemia can be checked three daysafter this procedure by transcardiac perfusion of a solution ofparaformaldehyde (4% in PBS).

After removing the brain, it was placed in fixative containing 10%sucrose, and cryostatic sections (20 nm) fixed with cresyl violet wereexamined under the optical microscope. Acetyl L-carnitine (50 mg/kg), orthe carnitine mixture (50 mg/kg of a mixture of acetyl L-carnitine,propionyl L-carnitine, and isovaleryl L-carnitine in a 1:1 weight ratioto one another), or α-lipoic acid (20 mg/kg) were administeredintravenously 5 minutes after the endothelin injection.

The volume of the infarcted area was calculated according to the methoddescribed by Park (Park C. K., Anns. Neurol., 20-150, 1989). The resultsof these tests demonstrate that acetyl L-carnitine, the carnitinemixture, and α-lipoic acid are all capable of reducing the ischaemicarea, but, surprisingly, the greatest and most significant result can beobtained with a combination of these products and, in particular, with acombination of acetyl L-carnitine and α-lipoic acid.

TABLE 1 Extent of ischaemia (volume in mm³) due to occlusion of the MCA(percentage reduction of volume compared to controls) Volume (mm³)Acetyl L-carnitine 25.6 ± 1.5 α-Lipoic acid 34.5 ± 2.1 Carnitine mixture35.8 ± 3.1 Acetyl L-carnitine + α-lipoic acid 85.3 ± 4.4 Carnitinemixture + α-lipoic acid 80.5 ± 6.1

Experimental Diabetic Hyperglycaemia Tests

Hyperglycaemia, whether through the formation of protein glycosylationproducts (AGEs) or through metabolic hypoxia, is one of the underlyingfactors responsible for diabetic disease and particularly for diabeticneuropathy.

Controlling serum glucose is therefore one of the most important meansof preventing diseases related to diabetes. In these tests, experimentaldiabetes was induced in rats, and tests were then performed to establishwhether the induced hyperglycaemia could be reduced by theadministration of acetyl L-carnitine, or carnitine mixture, or α-lipoicacid, or combinations of these products. The hyperglycaemia was inducedby subcutaneous injection of alloxan (100 mg/kg) in the rat, and thoserats were considered hyperglycaemia which presented serum glucose levelsabove 450 mg/dl seven days after the alloxan injection.

Treatment with the test substances was given orally for a period ofthree weeks. At the end of this period, serum glucose was measured inthe various groups of rats, both hyperglycaemic and treated.

The results obtained demonstrate that both carnitines and α-lipoic acidalone are capable of only slightly lowering the high initial serumglucose values, but the most significant result is that which appearsafter administration of carnitines in admixture with α-lipoic acid. Inthis case, particularly with the combination of acetyl L-carnitine andα-lipoic acid, there is a marked synergistic action of the two productswhich are capable of bringing serum glucose values down almost tonormal.

TABLE 2 Experimental hyperglycaemia tests in the rat Serum glucose(mg/dl) Treatment Initial After 21 days Controls 456.8 ± 26 505.5 ± 31Acetyl L-carnitine 509.5 ± 28 405.9 ± 25 Carnitine mixture 490.2 ± 32410.5 ± 35 α-lipoic acid 502.8 ± 36 360.4 ± 30 Acetyl L-carnitine +α-lipoic acid 489.6 ± 40 145.5 ± 21 Carnitine mixture + α-lipoic acid505.5 ± 39 170.5 ± 36 Acetyl L-carnitine = 200 mg/kg Carnitine mixture =acetyl L-carnitine + propionyl L-carnitine + isovaleryl L-carnitine in a1.1 ratio to one another α-Lipoic acid = 50 mg/kg

Test of Sorbitol Content in Ocular Lens and Sciatic Nerve in theDiabetic Rat

One of the most frequent causes of lesions induced by diabetichyperglycaemia and of the ocular or peripheral nervous diseasesassociated with it consists in the intracellular accumulation ofsorbitol, with consequent reduction of osmotic capacity and cellintegrity.

These tests were conducted in a group of rats in which diabetes wasinduced by means of the intravenous administration of 50 mg/kg ofstreptozotocin. One week after injection, serum glucose was tested andthose rats were considered diabetic which presented serum glucose valuesabove 450 mg/dl. These animals then received intraperitoneal injections,for eight consecutive days, of acetyl L-carnitine (100 mg/kg), orcarnitine mixture (acetyl L-carnitine+propionyl L-carnitine+isovalerylL-carnitine in a 1:1 weight ratio to one another) (100 mg/kg), or(x-lipoic acid (25 mg/kg), either alone or in various combinations.

After eight days of treatment, after suitable isolation, the sorbitolconcentration present in both the sciatic nerve and the ocular lens ofdiabetic rats was measured before and after the treatment administered.The sorbitol concentration appeared to be decreased in all animalstreated, but the most marked reduction was that detectable in theanimals treated with the combination of α-lipoic acid and carnitines,and the lowest values were recorded in the group treated with thecombination of acetyl L-carnitine and α-lipoic acid .

The results of these tests also show a surprising degree of synergisticpotentiation activity between α-lipoic acid and carnitine p.

TABLE 3 Sorbitol content in ocular lens and sciatic nerve in thediabetic rat Sorbitol (nmol/mg) Treatment Lens Sciatic nerve Controls 0.44 ± 0.06 0.078 ± 0.008 Diabetics 40.2 ± 3.9 1.85 ± 0.21 AcetylL-carnitine 32.7 ± 2.5 1.15 ± 0.11 Carnitine mixture 30.5 ± 2.9 1.05 ±0.09 α-lipoic acid 30.8 ± 3.2 1.08 ± 0.10 Acetyl L-carnitine + α-lipoicacid 14.7 ± 2.8 0.55 ± 0.08 Carnitine mixture + α-lipoic acid 16.4 ± 1.90.65 ± 0.07

Tests of Survival and Growth of Nerve Cells Treated with IGFc-ICarnitines and α-lipoic Acid

In view of the important role that insulin-like growth factor (IGF-I)plays in protecting the functional integrity of nerve cells,particularly against toxic lesion s such as those presenting in thecourse of diabetic diseases, we considered whether the activity of IGF-Ifavouring the growth and survival of brain cells was facilitated by thepresence in the culture medium of carnitines, or α-lipoic acid, or ofthese products in combination. To this end, brain cells of Wistar ratswere isolated according to the method described by Thanguipon(Thanguipon W., Dev. Brain Res., 11, 177, 1983) and were distributed onplates with a density of 3×10⁵/cm². To the culture medium was addedcytosine arabino-furanoside (10 mM) to prevent the replication ofnon-neuronal cells. After eight days, the cells were washed andmaintained in a culture in which the serum was replaced with BME (BasalEagles Medium, Life Technologies, Gaithersburg, Md.) containing a 5 mMKCl concentration. The test products were added directly to the serumthus prepared: IGF-I corresponding to 25 ng/ml acetyl L-carnitine (100ng/ml) or carnitine mixture (100 ng/ml) or α-lipoic acid, either aloneor in combination.

Cell survival and growth in culture were observed 24 hours afteraddition of the substances to the medium, on 35 mm disks in contact with10 ng/ml of fluorescein acetate according to the technique described byJones (Jones K. H., J. Histochem. Cytochem., 33, 77, 1985). The cellcount was done with a fluorescence microscope. The results of this testindicate that the growth-promoting effect of IGF-I on isolated braincells is significantly potentiated by the presence of carnitines andα-lipoic acid, which alone do not give rise to significant changes, andthat the greatest growth-potentiating effect is that induced whencarnitines, and particularly acetyl L-carnitine, are combined withα-lipoic acid.

TABLE 4 Tests of potentiation of IGF-1 enhancement of isolated braincell growth by acetyl L-carnitine, carnitine mixture, and α-lipoic acidCell growth Treatment (% vs controls) IGF-I 45 ± 2.8 Acetyl L-carnitine 5 ± 0.51 Carnitine mixture  9 ± 0.91 α-lipoic acid  5 ± 0.39 IGF-I +acetyl L-carnitine 65 ± 5.8 IGF-I + carnitine mixture 60 ± 3.5 IGF-I +α-lipoic acid 70 ± 6.1 IGF-I + acetyl L-carnitine + α-lipoic acid 98 ±7.3 IGF-I + carnitine mixture + α-lipoic acid 90 ± 6.9

Sciatic Nerve Regeneration Tests in Diabetic Rats

Rats with induced diabetes whose sciatic nerve has been cut presentinferior regenerative activity to that of normal rats.

These tests were conducted to investigate whether regeneration of thesciatic nerve in diabetic rats may be accelerated by treatment withacetyl L-carnitine, carnitine mixture, or α-lipoic acid, or combinationsof these products. The technique used in these tests is the onedescribed by Fernandez (Fernandez E., Int. J. Clin. Pharmacol. Res., 10,85, 1990).

Diabetes (serum glucose above 450 mg/dl) was induced in a group of ratsby subcutaneous injection of 100 mg/kg of alloxan. Acetyl L-carnitine,carnitine mixture and α-lipoic acid were administered with the diet insuch a way that the daily intake was 200 mg/kg of acetyl L-carnitine,200 mg/kg of carnitine mixture (acetyl L-carnitine+propionylL-carnitine+isovaleryl L-carnitine in a 1:1 weight ratio to one another)and 50 mg/kg of α-lipoic acid. The compounds were administered a weekbefore cutting the sciatic nerve and for thirty days after cutting.

The sciatic nerve was cut under anaesthesia and after exposing 1 cm ofit at the level of the sciatic foramen. The border of the lesion wasmarked with an epineural suture. Thirty days after cutting the nerve,the tissue of the tibial nerve, one of the main divisions of the sciaticnerve, was examined, after sacrificing the animals. Four cross-sectionsof the tibial nerve measuring approximately 4 mm in length were thussubjected to morphological and morphometric examination by means of asemiautomatic image analyser (Zeiss Videoplan Image Analyser).

The number of regenerating axons and their density per 100 nm² werecounted, as well as the degenerate elements. It thus proved possible todetect the diabetes-induced degeneration of the tibial nerve elements,which was corrected almost to the extent of restoring normal values bytreatment with acetyl L-carnitine, carnitine mixture, and α-lipoic acid.

The most evident results in terms of prevention of diabetic damage tonerve regeneration were those obtained with the administration of acetylL-carnitine or carnitine mixture in combination with α-lipoic acid, thusdemonstrating, in this test, too, a marked and unexpected synergism onthe part of the combination according to the invention.

TABLE 5 Number and density of tibial nerve degenerate elements aftercutting the sciatic nerve in diabetic rats Density Treatment Number (per100 nm²) Controls 965 ± 141 0.31 ± 0.04 Acetyl L-carnitine 560 ± 61 0.16± 0.02 Carnitine mixture 520 ± 55 0.14 ± 0.02 α-lipoic acid 590 ± 0.700.20 ± 0.04 Acetyl L-carnitine + α-lipoic acid 340 ± 0.41 0.10 ± 0.01Carnitine mixture + α-lipoic acid 360 ± 0.55 0.11 ± 0.02

Neuromuscular Conduction Tests

One of the most evident abnormalities in peripheral neuropathies andparticularly in diabetic neuropathy is the slowing down of neuromuscularconduction which is reflected in changes in motor activity.

In these tests, we induced experimental diabetes in rats byintravenously injecting the experimental animals (rats with a meanweight of 300 g) with 50 mg/kg of streptozotocin. In the animals withinduced diabetes (serum glucose above 450 mg/dl) the neuromuscularconduction velocity (NMCN7) was measured. To this end, the sciatic nervewas isolated (2 cm length) and the soleus muscle was separated from thegastrocnemius and its distal tendon cut and connected up to an isometrictransducer which recorded the muscular contraction force (MCF). Themuscle was stimulated via the sciatic nerve by means of two electrodesinserted at a distance of 10 mm from the nerve and connected up to astimulator.

A bipolar electrode was placed in the distal end of the muscle fordisplaying the electromyogram via an oscilloscope.

The NMCV was measured in m/sec, dividing the distance between thestimulation electrodes by the mean difference in latency between thestart of the ECG potentials evoked in the two sites. The MCF wasexpressed in mm.

TABLE 6 Neuromuscular conduction tests in the diabetic rat (after 4weeks) NMCV MCF Treatment (m/sec) (mm) Controls 42.2 ± 2.4 49.3 ± 3.1Diabetics 34.5 ± 2.1 34.6 ± 2.9 Diabetics + acetyl L-carnitine 38.5 ±1.9 40.6 ± 3.4 Diabetics + carnitine mixture 39.9 ± 2.1 41.2 ± 2.7Diabetics + α-lipoic acid 40.1 ± 1.5 41.9 ± 3.3 Diabetics + acetylL-carnitine + α-lipoic acid 43.4 ± 2.4 48.9 ± 3.9 Diabetics + carnitinemixture + α-lipoic acid 42.0 ± 3.1 47.5 ± 4.1

Motor Co-ordination Abnormality Test

These tests were conducted in “wobbler mice”, that is to say animalsthat present an unsteady, staggering gait, an abnormal position of thepaws and a reduced speed of movement. These abnormalities are related toprogressive atrophy of the motoneurons and musculo-cutaneous nervefibres, particularly as affecting the anterior limbs. The tests wereconducted according to the procedure proposed by Mitsumoto (MitsumotoH., Annal. Neurol., 36, 14, 1994). After diagnosis the wobbler mice weretreated orally for twenty days consecutively with acetyl L-carnitine(200 mg/kg), or with carnitine mixture (200 mg/kg), or with α-lipoicacid (50 mg/kg), or with these products in various combinations. Theexamination was performed by evaluating, in the treated animals versuscontrols, the time that each animal holds on to the edge of an inclinedplatform (holding time) and also the time it takes to run a distance of10 cm (running time).

The results of these tests indicate that the treatment of these animalswith acetyl L-carnitine, carnitine mixture and α-lipoic acid improvesboth holding time and running time as compared to controls, but alsothat the best effects are obtained with the administration of theseproducts in combination and in particular by the combination of acetylL-carnitine and α-lipoic acid. In these tests, too, there is a marked,unexpected synergistic potentiating effect of carnitines and α-lipoicacid.

TABLE 7 Tests of percentage increase in running time Controls 55 ± 4.5Acetyl L-carnitine 35 ± 3.2 Carnitine mixture 38 ± 4.1 α-lipoic acid 40± 3.9 Acetyl L-carnitine + α-lipoic acid 20 ± 1.9 Carnitine mixture +α-lipoic acid 26 ± 2.1

TABLE 8 Tests of percentage increase in holding time Controls 70 ± 5.5Acetyl L-carnitine 55 ± 4.6 Carnitine mixture 60 ± 3.8 α-lipoic acid 50± 5.9 Acetyl L-carnitine + α-lipoic acid 25 ± 3.2 Carnitine mixture +α-lipoic acid 30 ± 2.8

Tests of Cisplatin-induced Sensory Neuronal Lesions

The prolonged administration of cisplatin to experimental animals iscapable of causing lesions at the level of the sensory neurons and ofcausing marked abnormalities of propioceptive perception.

In these tests, we evaluated the protective effect exerted by theadministration, for seven consecutive days, of 300 mg/kg of acetylL-carnitine orally, or 300 mg/kg of carnitine mixture (acetylL-carnitine+propionyl L-carnitine+isovaleryl L-carnitine in a 1:1 weightratio to one another), or of 50 mg/kg of α-lipoic acid, or of theseproducts in various combinations on the toxicity induced by thesubcutaneous injection of 10 mg/kg of cisplatin for seven daysconsecutively.

The proprioceptive sensory perception abnormalities induced by cisplatinin the mouse were evaluated by means of the rotarod test (Apfel, S. C.,Ann. Neurol., 29, 89, 1991).

The results obtained in these tests demonstrate that, whereas cisplatincauses a substantial reduction in equilibrium time in cisplatin-treatedanimals as compared to control animals and reductions of the same orderin the animals treated with acetyl L-carnitine or α-lipoic acid alone,the group of animals treated with the combination of acetyl L-carnitineand α-lipoic acid, on the other hand, show an equilibrium capabilitypractically identical to that of the animals not subjected to cisplatinintoxication. In these tests, too, there is a marked, synergistic effectof carnitines and α-lipoic acid.

TABLE 9 Tests of neurosensory abnormalities induced by cisplatin(rotarod test) Equilibrium time Cisplatin Treatment (in seconds) —Controls 14.8 ± 1.4  Cisplatin — 8.4 ± 0.8 Cisplatin acetyl L-carnitine9.5 ± 0.6 Cisplatin carnitine mixture 8.9 ± 0.6 Cisplatin α-Lipoic acid9.9 ± 0.8 Cisplatin acetyl L-carnitine + α-lipoic acid 14.4 ± 1.8 Cisplatin carnitine mixture + α-lipoic acid 13.8 ± 2.1 

The tests performed to evaluate the activity that the new composition iscapable of exerting fully justify the innovative nature of the inventionitself and demonstrate above all the unexpected and surprisingsynergistic effect which its components are capable of inducing whenused in combination.

On the basis of the synergistic interaction of its components, thecomposition according to the invention described herein is suitable forpreventing toxic and metabolic damage which gives rise to neuronallesions of an acute or chronic nature. In particular, it can be used inthe treatment of toxic neuropathies, especially diabetic peripheralneuropathies.

In view of its antioxidant capability, this composition is alsoindicated in the prevention or treatment of abnormalities of toxic oranoxic origin related to the release of free radicals in the brain,liver, heart or other organs and tissues.

Furthermore, in view of the ability of the composition to promote theaction of IGF-I, pathological abnormalities related to ageing, such asneuro- degenerative disorders, may also obtain satisfactory benefit fromits use.

Illustrative, non-limiting examples of formulations according to theinvention are reported hereinbelow.

1) Acetyl L-carnitine mg 500 α-lipoic acid mg 50 2) Carnitine mixture mg500 (acetyl L-carnitine, propionyl L-carnitine, isovaleryl L-carnitinein identical weight amounts) α-lipoic acid mg 50 3) Acetyl L-carnitinemg 250 α-lipoic acid mg 25 4) Carnitine mixture mg 250 (acetylL-carnitine, propionyl L-carnitine, isovaleryl L-carnitine in identicalweight amounts) α-lipoic acid mg 25 5) Acetyl L-carnitine mg 1 α-lipoicacid mg 100 6) Acetyl L-carnitine mg 250 α-lipoic acid mg 25 Seleniummethionine μg 50 Zinc glycinate mg 10 Magnesium stereate mg 20 Taurinemg 50 Vit. E mg 10 CoQ₁₀ mg 10 β-carotene mg 10 Vit. C mg 30

What is meant by pharmacologically acceptable salt of L-carnitine oralkanoyl L-carnitine is any salt of these active ingredients with anacid that does not give rise to unwanted toxic or side effects. Theseacids are well known to pharmacy experts.

Non-limiting examples of suitable salts are the following: chloride;bromide; iodide; aspartate, acid aspartate; citrate, acid citrate;tartrate; phosphate, acid phosphate; fumarate; acid fumarate;glycerophosphate; glucose phosphate; lactate; maleate, acid maleate;orotate; oxalate, acid oxalate; sulphate, acid sulphate,trichloroacetate, trifluoroacetate and methanesulphonate.

A list of FDA-approved pharmacologically acceptable salts is given inInt. J. of Pharm. 33, (1986), 201—217; this latter publication isincorporated herein by reference.

The composition according to the invention may also comprise vitamins,coenzymes, minerals substances and antioxidants.

Appropriate excipients to be used to prepare the compositions havingregards to the specific route of administration, will be apparent to thepharmacy and food industry experts.

What is claimed is:
 1. A combination composition which comprises: (a)acetyl L-carnitine or a pharmacologically acceptable salt thereof; and(b) α-lipoic acid in a synergistically effective weight ratio.
 2. Thecomposition of claim 1, wherein ingredient (a) further comprises acarnitine selected from the group consisting of L-carnitine, propionylL-carnitine, valeryl L-carnitine, isovaleryl L-carnitine and theirphramacologially acceptable salts or mixtures thereof.
 3. Thecomposition of claim 1 or 2 wherein the weight ratio (a):(b) is from100:1 to 1:10.
 4. The composition of claim 1 wherein a pharmacologicallyacceptable salt of acetyl L-carnitine is selected from the groupconsisting of chloride, bromide, iodide, aspartate, acid aspartate,citrate, acid citrate, tartrate, phosphate, acid phosphate, fumarate,acid fumarate, glycerophosphate, glucose phosphate, lactate, maleate,acid maleate, orotate, acid oxalate, sulphate, acid sulphate,trichloroacetate, trifluoroacetate and methane sulfonate.
 5. Thecomposition of claim 2 wherein the carnitine selected is thepharmacologically acceptable salt of L-carnitine or alkanoyl L-carnitineselected from the group consisting of chloride, bromide, iodide,aspartate, acid aspartate, citrate, acid citrate, tartrate, phosphate,acid phosphate, fumarate, acid fumarate, glycerophosphate, glucosephosphate, lactate, maleate, acid maleate, orotate, acid oxalate,sulphate, acid sulphate, trichloroacetate, trifluoroacetate and methanesulphonate.
 6. The composition of claim 1 or 2 which further comprisesvitamins, coenzymes, mineral substances or antioxidants.
 7. Thecomposition of claim 1 or 2 in an orally administrable form as a dietarysupplement.
 8. The composition of claim 1 or 2 in an orally,parenterally, rectally or transdermally administrable form as amedicament.
 9. The composition of claim 7 in solid, semi-solid or liquidform.
 10. The composition of claim 9 in the form of tablets, lozenges,pills, capsules, granulates or syrups.
 11. The composition of claim 10in the form of tablets, lozenges, pills, capsules, granulates, syrups,injection or drops.
 12. A method of preventing tissue damage broughtabout by the presence of free radicals due to environmental pollution;for preventing brain or myocardial lesions induced by free radicalsfollowing cerebral or myocardial ischaemia and attendant reperfusion;for preventing diabetic or toxic neuropathies; or metabolic disorders inglucose utilization, said method comprising administering to a subjectin need of same a composition which in combination comprises: (a) acetylL-carnitine or a pharmacologically acceptable salt thereof andoptionally also a carnitine selected from the group consisting ofL-carnitine, propionyl L-carnitine, valeryl L-carnitine, isovalerylL-carnitine or their pharmacologically acceptable salts or mixturesthereof and (b) α-lipoic acid.
 13. A method of treating a diseasebrought about by the presence of free radicals due to environmentalpollution; brain or myocardial lesions induced by free radicalsfollowing cerebral or myocardial ischaemia and attendant reperfusion;atherosclerosis lesions and tissue proliferative processes; diabetic ortoxic neuropathies; and of metabolic disorders in glucose utilization,said method comprising administering to a subject in need of same acomposition which in combination comprises: (a) acetyl L-carnitine or apharmacologically acceptable salt thereof and optionally also acarnitine selected from the group consisting of L-carnitine, propionylL-carnitine, valeryl L-carnitine, isovaleryl L-carnitine or theirpharmacologically acceptable salts or mixtures thereof and (b) α-lipoicacid.
 14. The method of claim 12 wherein the weight ratio (a):(b) isfrom 100:1 to 1:10.
 15. The method of claim 13 wherein the weight ratio(a):(b) is from 100:1 to 1:10.